Glucocorticoids (GCs) are a class of steroid hormones that regulate numerous physiological events in the human body. Clinically, glucocorticoids are used for anti-inflammatory and immunosuppressive actions via binding with glucocorticoid receptors (GRs). Emerging evidence has also indicated that inappropriate GC and GR levels are detrimental for brain development and eventually lead to severe neurological diseases. However, the roles of GC/GR signaling in brain development are not fully understood. Here, we showed that stable GR expression levels were critical for brain development, because both GR knockdown and overexpression severely impaired neuronal migration. Further studies showed that the multipolar–bipolar transition and leading process development were interrupted in GR-knockdown and GR-overexpressing neurons. To elucidate the underlying mechanism, we screened the protein levels of downstream molecules and identified RhoA as a factor negatively regulated by the GR. Restoration of the RhoA protein level partially rescued the neuronal migration defects in the GR-knockdown and GR-overexpressing neurons, indicating that RhoA played a major role in GR-mediated neuronal migration. These data suggest that an appropriate level of GC/GR signaling is essential for precise control of neuronal migration.
Huali Yu, Ye Guo, Yang Zhao, Feng Zhou, Kehan Zhao, Mayuqing Li, Junxiong Wen, Zixuan He, Xiaojuan Zhu and Xiaoxiao He
Ioannis Simitsidellis, Arantza Esnal Zufiaurre, Olympia Kelepouri, Elizabeth O’Flaherty, Douglas A Gibson and Philippa T Saunders
Selective androgen receptor modulators (SARMs) have been proposed as therapeutics for women suffering from breast cancer, muscle wasting or urinary incontinence. The androgen receptor (AR) is expressed in the uterus but the impact of SARMs on the function of this organ is unknown. We used a mouse model to compare the impact of SARMs (GTx-007/Andarine, GTx-024/Enobosarm), Danazol (a synthetic androstane steroid) and dihydrotestosterone (DHT) on tissue architecture, cell proliferation and gene expression. Ovariectomised mice were treated daily for 7 days with compound or vehicle control (VC). Uterine morphometric characteristics were quantified using high-throughput image analysis (StrataQuest; TissueGnostics), protein and gene expression were evaluated by immunohistochemistry and RT-qPCR, respectively. Treatment with GTx-024, Danazol or DHT induced significant increases in body weight, uterine weight and the surface area of the endometrial stromal and epithelial compartments compared to VC. Treatment with GTx-007 had no impact on these parameters. GTx-024, Danazol and DHT all significantly increased the percentage of Ki67-positive cells in the stroma, but only GTx-024 had an impact on epithelial cell proliferation. GTx-007 significantly increased uterine expression of Wnt4 and Wnt7a, whereas GTx-024 and Danazol decreased their expression. In summary, the impact of GTx-024 and Danazol on uterine cells mirrored that of DHT, whereas GTx-007 had minimal impact on the tested parameters. This study has identified endpoints that have revealed differences in the effects of SARMs on uterine tissue and provides a template for preclinical studies comparing the impact of compounds targeting the AR on endometrial function.
E J Agnew, A Garcia-Burgos, R V Richardson, H Manos, A J W Thomson, K Sooy, G Just, N Z M Homer, C M Moran, P J Brunton, G A Gray and K E Chapman
Endogenous glucocorticoid action is important in the structural and functional maturation of the fetal heart. In fetal mice, although glucocorticoid concentrations are extremely low before E14.5, glucocorticoid receptor (GR) is expressed in the heart from E10.5. To investigate whether activation of cardiac GR prior to E14.5 induces precocious fetal heart maturation, we administered dexamethasone in the drinking water of pregnant dams from E12.5 to E15.5. To test the direct effects of glucocorticoids upon the cardiovascular system we used SMGRKO mice, with Sm22-Cre-mediated disruption of GR in cardiomyocytes and vascular smooth muscle. Contrary to expectations, echocardiography showed no advancement of functional maturation of the fetal heart. Moreover, litter size was decreased 2 days following cessation of antenatal glucocorticoid exposure, irrespective of fetal genotype. The myocardial performance index and E/A wave ratio, markers of fetal heart maturation, were not significantly affected by dexamethasone treatment in either genotype. Dexamethasone treatment transiently decreased the myocardial deceleration index (MDI; a marker of diastolic function), in control fetuses at E15.5, with recovery by E17.5, 2 days after cessation of treatment. MDI was lower in SMGRKO than in control fetuses and was unaffected by dexamethasone. The transient decrease in MDI was associated with repression of cardiac GR in control fetuses following dexamethasone treatment. Measurement of glucocorticoid levels in fetal tissue and hypothalamic corticotropin-releasing hormone (Crh) mRNA levels suggest complex and differential effects of dexamethasone treatment upon the hypothalamic–pituitary–adrenal axis between genotypes. These data suggest potentially detrimental and direct effects of antenatal glucocorticoid treatment upon fetal heart function.
A Edlund, M Barghouth, M Hühn, M Abels, J S E Esguerra, I G Mollet, E Svedin, A Wendt, E Renström, E Zhang, N Wierup, B J Scholte, M Flodström-Tullberg and L Eliasson
Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine alpha and beta cell function in the Cftr tm1 EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild type (WT) mice aged 7–10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in (1) reduced number of docked insulin granules and reduced exocytosis and (2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD.
Thomas Nicholson, Chris Church, Kostas Tsintzas, Robert Jones, Leigh Breen, Edward T Davis, David J Baker and Simon W Jones
Adipokines have emerged as central mediators of insulin sensitivity and metabolism, in part due to the known association of obesity with metabolic syndrome disorders such as type 2 diabetes. Recent studies in rodents have identified the novel adipokine vaspin as playing a protective role in inflammatory metabolic diseases by functioning as a promoter of insulin sensitivity during metabolic stress. However, at present the skeletal muscle and adipose tissue expression of vaspin in humans is poorly characterised. Furthermore, the functional role of vaspin in skeletal muscle insulin sensitivity has not been studied. Since skeletal muscle is the major tissue for insulin-stimulated glucose uptake, understanding the functional role of vaspin in human muscle insulin signalling is critical in determining its role in glucose homeostasis. The objective of this study was to profile the skeletal muscle and subcutaneous adipose tissue expression of vaspin in humans of varying adiposity, and to determine the functional role of vaspin in mediating insulin signalling and glucose uptake in human skeletal muscle. Our data shows that vaspin is secreted from both human subcutaneous adipose tissue and skeletal muscle, and is more highly expressed in obese older individuals compared to lean older individuals. Furthermore, we demonstrate that vaspin induces activation of the PI3K/AKT axis, independent of insulin receptor activation, promotes GLUT4 expression and translocation and sensitises older obese human skeletal muscle to insulin-mediated glucose uptake.
Jin Yu, Yuhuan Liu, Danying Zhang, Dongxia Zhai, Linyi Song, Zailong Cai and Chaoqin Yu
High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ELISA. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, baicalin decreased the testosterone concentrations in a dose- and time-dependent manner in NCI-H295R cells. GEP revealed that 3β-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity and decreased the expression of HSD3B2 in ovarian. In summary, our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin will potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.
K E Lines, P J Newey, C J Yates, M Stevenson, R Dyar, G V Walls, M R Bowl and R V Thakker
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1 +/ − mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1 +/ − mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.
Lesley A Hill, Dimitra A Vassiliadi, Ioanna Dimopoulou, Anna J Anderson, Luke D Boyle, Alixe H M Kilgour, Roland H Stimson, Yoan Machado, Christopher M Overall, Brian R Walker, John G Lewis and Geoffrey L Hammond
Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.
Vikte Lionikaite, Karin L Gustafsson, Anna Westerlund, Sara H Windahl, Antti Koskela, Juha Tuukkanen, Helena Johansson, Claes Ohlsson, H Herschel Conaway, Petra Henning and Ulf H Lerner
Excess vitamin A has been associated with decreased cortical bone thickness and increased fracture risk. While most studies in rodents have employed high dosages of vitamin A for short periods of time, we investigated the bone phenotype in mice after longer exposure to more clinically relevant doses. For 1, 4 and 10 weeks, mice were fed a control diet (4.5 µg retinyl acetate/g chow), a diet modeled from the human upper tolerable limit (UTL; 20 µg retinyl acetate/g chow) and a diet three times UTL (supplemented; 60 µg retinyl acetate/g chow). Time-dependent decreases in periosteal circumference and bone mineral content were noted with the supplemented dose. These reductions in cortical bone resulted in a significant time-dependent decrease of predicted strength and a non-significant trend toward reduced bone strength as analyzed by three-point bending. Trabecular bone in tibiae and vertebrae remained unaffected when vitamin A was increased in the diet. Dynamic histomorphometry demonstrated that bone formation was substantially decreased after 1 week of treatment at the periosteal site with the supplemental dose. Increasing amount of vitamin A decreased endocortical circumference, resulting in decreased marrow area, a response associated with enhanced endocortical bone formation. In the presence of bisphosphonate, vitamin A had no effect on cortical bone, suggesting that osteoclasts are important, even if effects on bone resorption were not detected by osteoclast counting, genes in cortical bone or analysis of serum TRAP5b and CTX. In conclusion, our results indicate that even clinically relevant doses of vitamin A have a negative impact on the amount of cortical bone.
Jin-Ran Chen, Oxana P Lazarenko, Haijun Zhao, Alexander W Alund and Kartik Shankar
Intrauterine or early postnatal high-fat diet (HFD) has substantial influences on adult offspring health; however, studies of HFD-induced maternal obesity on regulation of adult offspring bone formation are sparse. Here, we investigated the effects of HFD-induced maternal obesity on both fetal and adult offspring skeletal development. We found that HFD-induced maternal obesity significantly decreased fetal skeletal development, but enhanced fetal osteoblastic cell senescence signaling and significantly increased the expression of inflammatory factors of the senescence-associated secretory phenotype (SASP) in osteo-progenitors. It was found that p300/CBP activation led to H3K27 acetylation to increase the expression of senescence-related genes and PPARγ in embryonic mouse osteogenic calvarial cells from HFD obese dams. These results were recapitulated in human umbilical cord mesenchymal stem cells (UC MSCs) isolated from offspring of pregnant obese and lean mothers following delivery. Regardless of postnatal HFD challenge, adult offspring from HFD obese dams showed significantly suppressed bone formation. Such early involution of bone formation of adult offspring from HFD obese dams may at least in part due to histone acetylation, i.e., epigenetic regulation of genes involved in cell senescence signaling in pre-osteoblasts from prenatal development. These findings indicate fetal pre-osteoblastic cell senescence signaling is epigenetically regulated by maternal obesity to repress bone formation in adult offspring in rodents and suggest that at least some of these effects may also manifest in humans.